Perhaps one of the most intriguing, yet least studied, aspects of intercellular transport in higher eukaryotes, from mammals to plants, is traffic of macromolecular complexes through intercellular cytoplasmic bridges between cells. These connections, termed tunneling nanotubes (TNTs) in mammals and plasmodesmata (Pd) in plants, are subverted by invading pathogens for their movement between the host cells. Whereas both mammalian and plant pathogens, e.g., prions and (potentially) HIV as well as most plant viruses, utilize cell-to-cell transport pathways, the first such capability was identified for plant viruses. Thus, viral transport via Pd represents a conceptual and mechanistic paradigm for intercellular traffic of macromolecules. We exploit Tobacco mosaic virus (TMV), whose Pd transport is mediated by its movement protein (MP), as a tool to study the regulatory mechanisms of Pd transport, focusing on two fundamental questions: (i) How does MP activate the host pathway for gating the Pd channel? And (ii) how does the host regulate this Pd-gating activity of MP? Our data suggest the presence of three regulatory mechanisms that involve MP: MP-induced activation of the cellular pathway for relaxation of a polysaccharide Pd sphincter, activation and deactivation of MP by phosphorylation, and down-regulation of MP by the host ubiquitin/proteasome system (UPS). These findings will be used to seek three objectives: Aim 1. Understand the mechanism by which MP gates Pd by modulating the polysaccharide sphincter. Our data identified a host cytoplasmic protein ANK that is recognized by MP and showed that the MP-ANK complexes accumulate at Pd and that the presence of ANK is required for MP-induced gating of Pd. We also showed that ANK interacts with ss-1, 3 glucanase (BG), an enzyme that degrades the polysaccharide Pd sphincter. We will test the hypothesis that MP redirects ANK from the cytoplasm to Pd, where ANK (or ANK-MP complexes) activates BG, that relaxes the Pd sphincter and elevates the Pd permeability. Aim 2. Understand the regulatory function of MP phosphorylation. We identified an ER- associated and Pd-associated protein kinases (ERPK and PdPK) that specifically phosphorylate MP, activating and deactivating its ability to gate Pd, respectively. We will explore the hypothesis that these PKs act as an On/Off switch of MP transport through Pd. Aim 3. Understand the role of the host UPS in down-regulation of MP. Our data show that challenge with pathogens induces expression of the plant defense-related F-box protein VBF. Among its pathogen-encoded substrates, VBF recognizes MP. We will test the hypothesis that VBF targets MP to proteasomal degradation via the SCFVBF pathway. Collectively, the expected outcomes of proposed experiments will define and characterize basic concepts and molecular mechanisms that underlies activation and deactivation of intercellular transport of macromolecular complexes in general, and pathogens in particular. PUBLIC HEALTH RELEVANCE: This proposal focuses on the mechanisms by which viral pathogens modulate permeability of the host intercellular connections for their spread, using as model system the movement protein (MP) of Tobacco mosaic virus which mediates viral transport through plasmodesmata (Pd). Specifically, we aim to determine (i) the mechanism by which MP gates Pd, (ii) the mechanism of MP activation and deactivation by phosphorylation On/Off switch, and (iii) the involvement of the host ubiquitin/proteasome system (UPS) in down-regulation of MP. In the general biological context, the significance of the proposed studies lies in (i) definition of the mechanistic concept-recruitment of the host factors to activate a pathway that removes the structural elements of the transport channel that restrict its permeability-employed by pathogens for spread between host cells via cytoplasmic intercellular channels, such as Pd or TNTs, and (ii) determination of molecular mechanisms that both the pathogen and the host use to control this subversion of intercellular transport.